Synthetic peptides and dna sequences for treatment of multiple sclerosis

ABSTRACT

Synthetic unaltered and altered peptides comprising sequences of at least one immunogenic epitope cluster (IEC) of at least one human autoantigen related to multiple sclerosis (MS) and at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N- and C-termini, are provided. The alteration is preferably by substituting 1 to 3 TCR contact residues by Ala. The autoantigen is preferably MOG, MBP, OSP, MOBP and PLP. Polypeptides comprising at least two such peptides of a sole autoantigen or at least one peptide of two different autoantigens, and synthetic genes encoding them, are also provided, as well as pharmaceutical compositions for treatment and diagnostic of MS.

FIELD OF THE INVENTION

The present invention relates to synthetic peptides and polypeptides and synthetic human genes useful for the treatment of multiple sclerosis as well as for diagnostic purposes.

Abbreviations: AL: altered ligand; APL: altered peptide ligand; CNS: central nervous system; EAE: experimental autoimmune encephalomyelitis; IEC: immunogenic epitopic cluster; MAG: myelin-associated glycoprotein; MBP: myelin basic protein; MOBP: myelin-oligodendrocytic basic protein; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; OSP: oligodendrocyte-specific protein;. PBL: peripheral blood lymphocytes; PLP: proteolipid protein; PNS: peripheral nervous system; TCR: T-cell receptor

BACKGROUND TO THE INVENTION

Autoimmune diseases result from the immune system's failure to maintain self-tolerance to antigen(s) in the affected organ. Over 40 systemic and organ-specific autoimmune diseases have been observed. Among the organ-specific autoimmune diseases are multiple sclerosis, myasthenia gravis, thyroiditis, insulin-dependent diabetes mellitus, rheumatoid arthritis and others. In spite of major and significant advances in molecular and cellular immunology in the last two decades, the molecular basis for self-tolerance and the mechanisms regulating it are still a major challenge in immunology, and autoimmune diseases remain a major medical problem. The immune-specific approaches to therapy of the disease, expected to be the most effective, have not yet yielded an effective therapy for any of the autoimmune diseases.

Accordingly, many other approaches have been investigated, some of which resulted in a limited success in decreasing the progression of the disease, such as the use of β-interferon and Copolymer 1 for treatment of multiple sclerosis, yet none of them cure the disease. Apparently, the major difficulty in devising immune specific approaches to therapy lies in the complexity of the autoimmune diseases, particularly with regard to the multiplicity of target antigens and because of the possibility that the primary target antigen(s) may be different in different patients, the difficulty in determining which of the possible target antigens is the primary target antigen for each patient, and against which of the possible epitopes on that protein the pathogenic autoimmune response is primarily directed. This is further complicated by the likely “spread of autoimmunity” as disease develops.

By way of example, multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS) characterized by neurological impairment of varying extent, results from demyelination, which is believed to result from an autoimmune response against myelin. A number of CNS myelin proteins have been postulated to be potential primary target antigens in MS on the basis of their ability to induce experimental autoimmune encephalomyelitis (EAE), a well-accepted animal model for MS, and detection of autoreactivity to these antigens in MS patients (reviewed in Kerlero de Rosbo and Ben-Nun, 1998, 1999; Kaye et al., 2000; Stevens et al., 1999; Zhong et al., 2000). Among these, myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG), the major proteins of CNS myelin, have been extensively studied (reviewed in Kerlero de Rosbo and Ben-Nun, 1998, 1999).

Activated CD4+ T cells specific for MBP or PLP are sufficient to cause EAE upon their transfer into naive syngeneic recipients, and potentially pathogenic T cells reactive against MBP or PLP have been demonstrated in MS (reviewed in Tuohy, 1994); however, comparable T cell responses to MBP or PLP were also detected in healthy individuals (reviewed in Tuohy, 1994). Thus, although specific responses to these myelin antigens are likely to be of importance in the course of the disease, they may not represent the primordial pathogenic response in MS. Consequently, in the search for antigenic specificities associated with MS, other myelin-specific, and also more recently non myelin-specific CNS antigens, have been investigated for their encephalitogenicity and/or for the presence of autoreactivity to these antigens in MS. Thus, low levels of T cell response to myelin-associated glycoprotein (MAG) and S100b, found in CNS and PNS tissues, have been observed both in MS patients and control individuals and reactivity to non nervous system-specific antigens such as heat shock proteins, transaldolase, and, to a lesser extent, 2′,3′-cyclic nucleotide 3′-phosphodiesterase, has been reported in MS (reviewed in Kerlero de Rosbo and Ben-Nun, 1998). However, none of these antigens have so far been demonstrated to be encephalitogenic, albeit T cells specific for MAG and S100b can cause CNS and PNS inflammation upon passive transfer into syngeneic mice with no clinical manifestations.

In view of the restricted localization of MS lesions to the CNS white matter, it is more likely that a primary target antigen in MS is CNS myelin-specific. Myelin proteins such as MOG, myelin-oligodendrocytic basic protein (MOBP) and oligodendrocyte-specific protein (OSP) are believed to be specific components of CNS myelin (Gardinier et al., 1992; Yamamoto et al., 1994; Bronstein et al., 1997). Our studies on the reactivity to MOG by PBL (peripheral blood lymphocytes) from patients with MS in the context of their reactivity to MBP, PLP and MAG have shown that a high proportion (50%) of MS patients react predominantly to MOG (Kerlero de Rosbo et al., 1997). Most importantly, reactivity to MOG by PBL from control individuals occurs far less frequently (Kerlero de Rosbo et al., 1997). These data, together with the demonstration of the encephalitogenic potential of MOG, strongly suggests that autoimmune reactivity to this CNS myelin-specific antigen plays an important role in the pathogenesis of MS.

Another important point emerged from our investigation of the reactivity by MS PBL to the different myelin antigens, MBP, PLP, MAG and MOG, concomitantly: 40% of the MS patients tested showed no reactivity to any of these myelin antigens. Among the several explanations which could account for this observation, one likely possibility is the involvement of autoimmune reactivity to myelin-specific antigen(s) other than MBP, PLP or MOG. In this context, we have studied the autoreactivity to MOBP, a recently uncovered CNS myelin-specific protein, which is apparently relatively abundant in CNS myelin. Our data yielded from two separate studies of the proliferative response to MOBP by PBL from MS patients and controls indicated that, out of the twenty-two patients tested overall, eleven reacted to one or several MOBP peptides whilst only four out of twenty controls tested overall reacted (Kaye et al., 2000). The demonstration by us and another laboratory that MOBP is also encephalitogenic, provides unequivocal evidence that the autoimmune reactivity observed in MS patients is potentially pathogenic and may play an important role in the pathogenesis of MS. We (Zhong et al., 2000) and another laboratory (Stevens et al., 1999) also recently demonstrated the strong encephalitogenic activity of another CNS myelin-specific protein, OSP, indicating that OSP may also be a potential target antigen for autoimmune demyelinating diseases such as MS.

A potential primary target antigen in MS could be defined as a CNS antigen which has an encephalitogenic potential, i.e. can cause EAE, and against which autoimmune reactivity can be detected in MS patients. In this context, MBP, PLP, MOG and now also MOBP can be considered potential primary target antigens, as autoreactivity against one of these antigens may play an important role in the initiation/progression of MS. In view of its high encephalitogenicity, the potential role of autoimmune responses to OSP in the pathogenesis of MS should also be considered. In contrast, autoimmune responses to other nonencephalitogenic CNS components, myelin-specific or non myelin-specific, which can be detected in MS, are more likely to represent secondary events resulting from “autoimmune spread” as a result of inflammation within CNS with ongoing disease. The multiplicity of potential primary target antigens in MS points to the complexity of the disease with regard to possible pathogenic processes involved, possible etiology of the disease, and most importantly, it imposes major difficulties in devising immune-specific therapeutic approaches to MS.

Thus, the major problems that must be addressed by immune-specific therapies for a given autoimmune disease include the multiplicity of potential primary target antigens with the possibility that the primary target antigens differ in different patients, and the recently acknowledged “spreading of autoimmunity” as disease develops. This phenomenon is described as the observation of variation in the active immunogenic epitopes with the progression of the disease. This results in the evolution of the primary T cell response focused on a particular self-antigen, towards the recruitment of T cells to multiple antigenic determinants on this or other potential target autoantigens within the affected organ (Tuohy et al., 1998; Kumar, 1998).

PCT International Publication WO 01/31037 of the present applicants discloses synthetic human target autoantigen genes comprising sequences coding for at least two immunogenic epitope clusters (hereinafter EEC) of autoantigen(s) related to a specific autoimmune disease, wherein said at least two IECs may be derived from a sole autoantigen or from at least two different autoantigens related to said autoimmune disease, and polypeptides encoded thereby, that can be used for the treatment and diagnosis of autoimmune diseases such as multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA) and others. Several synthetic human genes have been disclosed in said WO 01/31037, each gene comprising sequences coding for at least two IECs of autoantigen(s) related to a specific autoimmune disease such as MS, IDDM or RA, said synthetic gene being selected from:(i) a synthetic human target autoantigen gene (designated shTAG) comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to said autoimmune disease; and (ii) a synthetic human multitarget autoantigen gene (designated shMultiTAG) comprising nucleotide sequences coding for at least one IEC of at least two different autoantigens related to said autoimmune disease. WO 01/31037 further disclosed several synthetic polypeptides, each polypeptide comprising amino acid sequences of at least two IECs of autoantigens related to a specific autoimmune disease such as MS, IDDM or RA, said synthetic polypeptide being selected from: (i) a synthetic human polypeptide (designated shPEP) comprising amino acid sequences of at least two IECs of a sole autoantigen related to said autoimmune disease; and (ii) a synthetic human multitarget polypeptide (designated shMultiPEP) comprising amino acid sequences of at least one IEC of at least two different autoantigens related to said autoimmune disease.

In MS, the multiplicity of potentially pathogenic autoreactivities against the myelin components, MBP, PLP, MOG, MOBP, and OSP, which have been detected in different patients, suggest that the primary target antigen(s) and/or the major epitope(s) against which the dominant pathogenic autoreactivities are directed, may differ in different patients. Because neoreactivities are also likely to emerge, disease progression may be associated with multiple potentially pathogenic T cell autoreactivities. This imposes major difficulties for devising immune-specific approaches for therapy of MS.

Thus, effective immune-specific therapy would have to be tailored for each patient according to the antigenic and epitope specificities of the potentially pathogenic autoimmune T-cells detected in that patient, including novel T-cell specificities elicited as a result of “autoimmune spread” as disease progresses. An alternative and more generally applicable approach would ideally be, if all or most of relevant potentially pathogenic autoreactivities could be targeted concomitantly. This would allow immunomodulation of MS, irrespective of the antigenic primacy or dominance of the pathogenic autoimmune response in individual patients.

Several studies in EAE strongly suggest that neutralizing T cells specific for one epitope may not be a sufficiently effective therapeutic approach for disease associated with multiple pathogenic autoreactivities. Thus, in chronic EAE induced in SJL/J mice with whole PLP, tolerization with the major encephalitogenic peptide, PLP139-151, abrogated the primary acute phase, but not subsequent relapses related to autoimmune spread. The clinical severity of EAE induced in (PL/J×SJL/J) F₁ mice with a combination of MBP and MOG could be significantly reduced by tolerogenic administration of a combination of the immunodominant encephalitogenic epitopes within MBP Ac1-11 and MOG41-60 (Leadbetter et al., 1998). In contrast, MBPAc1-11, which suppresses MBP-induced EAE in these F₁ mice, had no effect on MOG-induced EAE and a marginal therapeutic effect on EAE induced by the MBP/MOG combination (Leadbetter et al., 1998), an observation most likely related to its specific suppressive effect on MBP-reactive T cells. Highly relevant to treatment of disease with multiple autoreactivities is the strong therapeutic effect on EAE of MP4, a chimeric fusion protein, comprising the whole long isoform of MBP (21.5 kDa MBP) and the hydrophilic domains of PLP (ΔPLP) (Elliott et al., 1996).

Tolerogenic administration of MP4 fully abrogated EAE actively induced with PLP139-151, as well as EAE adoptively transferred with a combination of encephalitogenic MBP- and PLP-specific T cells in SJL/J mice. In contrast, neither 21.5 kDa MBP nor ΔPLP injected individually had any such dramatic effect on passive EAE mediated by the combined T cell populations (Elliott et al., 1996). Taken together, these studies suggest that targeting the majority of relevant T cells may be required for optimal efficacy of immune-specific therapy in disease associated with pathogenic T cell reactivities against more than one antigen/epitope.

In summary, the state of the art has convincingly demonstrated that, while EAE resulting from autoreactivity to a single autoantigen can be effectively suppressed by neutralization of the relevant T-cells via tolerogenic administration of the relevant epitope, a single epitope is not effective in suppressing EAE associated with multiple pathogenic autoreactivities. In contrast, targeting all relevant T-cells concomitantly can result in full abrogation of disease.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a synthetic peptide comprising amino acid sequences of at least one IEC of at least one human autoantigen related to MS, said synthetic peptide being selected from:

-   -   (i) an unaltered synthetic peptide comprising at least one         nonameric core sequence which fits into the MS-relevant         HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N-         and C-termini, the resulting IEC being capable of stimulating         human T cells, wherein the peptides are those of SEQ ID Nos: 10         and 22-47; and     -   (ii) an altered synthetic peptide comprising at least one         nonameric core sequence which fits into the MS-relevant         HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its         N-and C-termini, in which sequence 1 to 3 T-cell receptor (TCR)         contact amino acid residues are substituted by a suitable amino         acid such as Ala, the resulting IEC altered in the TCR residue         being capable of immunomodulating the potentially pathogenic         T-cell response against the epitope without risk of         exacerbation, excluding altered peptides derived from the MBP         83-99 and PLP 139-151 sequences.

The human autoantigen related to MS is preferably MOG, MBP, OSP, MOBP, PLP, and MAG.

Examples of unaltered peptides according to the invention are the MOG peptides of SEQ ID NOs: 22-25 (FIG. 1), the MBP peptides of SEQ ID NOs: 26-30 (FIG. 2), the OSP peptides of SEQ ID NOs: 10, 31-35 (FIG. 3), the MOBP peptides of SEQ ID NOs: 36-39 (FIG. 4), and PLP peptides of SEQ ID NOs: 40-47 (FIG. 5).

Examples of altered peptides according to the invention are the MOG-AL peptides of SEQ ID NOs: 48-53 (FIG. 15), the MBP-AL peptides of SEQ ID NOs: 54-61 (FIG. 16), the OSP-AL peptides of SEQ ID NOs: 62-69 (FIG. 17), the MOBP-AL peptides of SEQ ID NOs: 70-73 (FIG. 18), and the PLP-AL peptides of SEQ ID NOs: 74-86 (FIG. 19).

In another aspect, the present invention provides a synthetic gene comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS or coding for at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said IECs have amino acid sequences selected from the sequences of the unaltered and/or altered peptides of the invention, particularly those of SEQ ID NOs: 10 and 22-86.

In a further aspect, the present invention polypeptides that are the protein products of the synthetic genes of the invention and comprise amino acid sequences of at least two IECs of a sole autoantigen related to MS or at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said IECs have amino acid sequences selected from the sequences of the unaltered and/or altered peptides of the invention, particularly those of SEQ ID NOs: 10 and 22-86.

In still another aspect, the present invention provides pharmaceutical compositions comprising a mixture of at least two of the unaltered or altered peptides of the invention, or at least one polypeptide or a synthetic gene of the invention for treatment or diagnostic of MS.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a scheme of the herein designated shMOG gene.

FIG. 2 depicts a scheme of the herein designated shMBP gene.

FIG. 3 depicts a scheme of the herein designated shOSP gene.

FIG. 4 depicts a scheme of the herein designated shMOBP gene.

FIG. 5 depicts a scheme of the herein designated shPLP gene.

FIG. 6 depicts a scheme of the herein designated Y-MSPe gene.

FIG. 7 depicts a scheme of the herein designated ΔY-MSPe gene.

FIG. 8 shows that MOG altered peptide ligands (APLs) substituted at crucial T-cell receptor (TCR) contact residues 41 and 44 suppress EAE induced with peptide MOG 37-52. I and M indicate incidence of disease and mortality in the group, respectively.

FIGS. 9A-9B show that inhibition of EAE associated with multiple pathogenic autoreactivities requires multi-epitope-directed immunomodulation. 9A. Multiantigen/epitope reactivity of Y-MSPb-reactive line T cells. 9B. EAE associated with multiple pathogenic autoreactivities induced by Y-MSPb-reactive line T-cells (2×10−⁶ cells, i.v., on day 0) is fully abrogated by tolerogenic administration of Y-MSPb in PBS given daily intraperitoneally (i.p.) from the day of T-cell transfer. (SI=stimulation index).

FIG. 10 shows the MOG peptides of SEQ ID NOs: 1-4 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of MOG. Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.

FIG. 11 shows the MBP peptides of SEQ ID NOs: 5-7 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of MBP. Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.

FIG. 12 shows the OSP peptides of SEQ ID NOs: 8-13 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of OSP. Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.

FIG. 13 shows the MOBP peptides of SEQ ID NOs: 14-16 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of MOBP. Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.

FIG. 14 shows the PLP peptides of SEQ ID NOs: 17-21 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of PLP. Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.

FIG. 15 depicts a scheme of the herein designated shMOG-AL gene.

FIG. 16 depicts a scheme of the herein designated shMBP-AL gene.

FIG. 17 depicts a scheme of the herein designated shOSP-AL gene.

FIG. 18 depicts a scheme of the herein designated shMOBP-AL gene.

FIG. 19 depicts a scheme of the herein designated shPLP-AL gene.

FIG. 20 depicts a scheme of the herein designated Y-MSP-AL gene.

FIG. 21 depicts a scheme of the herein designated ΔY-MSP-AL gene.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Within the context of the specification the following definitions will be used: “Autoantigen” refers to the self-molecules (proteins) recognized as potential target antigens in an autoimmune disease. “Epitope” refers to an antigenic determinant of the autoantigen. “Immunogenic epitopic clusters (IEC's)” is used herein to refer to an epitope or collection of epitopes within a region of an autoantigen. The cluster may include one or more flanking, overlapping epitopes or such epitopes in tandem with one another. “Immunogenic (epitopic) cluster coding region” is used to refer to the nucleotide sequence that encodes for an IEC. “Immunogenic” is used herein to refer to the ability of an epitope to initiate an immune response. “Immunomodulatory” is used to refer to the ability of an IEC to modulate, regulate, control or antagonize an autoantigenic induction of an immune response in an appropriate animal model.

B. The Autoantigens for MS

For MS, several autoantigens including MBP, PLP, MAG, MOG, MOBP and OSP have been proposed as being potential targets for the primary autoimmune attack, by an assessment of peripheral blood lymphocytes (PBLs) of MS patients (Kerlero de Rosbo and Ben-Nun, 1998; Kaye et al., 2000) and/or by their ability to invoke a disease-related immunogenic response in the relevant animal model (Kerlero de Rosbo and Ben-Nun, 1999; Kaye et al., 2000; Stevens et al., 1999; Zhong et al., 2000).

C. The Immunogenic Epitopic Clusters (IECs) of the Autoantigens for MS

C1. Unaltered Peptides

The selection of the IECs of the identified potential target autoantigens related to MS is based on experimental identification of the epitopes most frequently recognized in patients, as assayed by reactivity to overlapping peptides of the relevant autoantigen, and/or on determination of the preferred binding mode of regions of the molecule to HLA associated with MS as predicted by computer modeling, preferably confirmed by binding assays and/or experimental data obtained in HLA-transgenic mice. The following Table 1 summarizes IECs which have been experimentally identified or predicted to be potential epitopes for MS, as described in WO 01/31037: TABLE 1 Autoimmune Auto- disease antigen Location of immunogenic cluster Multiple sclerosis MOG 34-56, 67-114, 3-27, 205-215 MBP 84-111, 141-168, 12-42 OSP 42-73, 98-110, 187-206, 21-34, 130-146 MOBP 15-33, 55-90, 156-172 PLP 103-150, 177-203, 218-240, 38-52, 264-276

In the present invention, computer modeling of the binding mode of the selected epitope clusters to HLA-DR/DQ molecules most prominently associated with MS, was used to predict for each epitope cluster the nonameric core sequences with preferred binding mode to these HLA-DR/DQ and which were seen to fit into the MHC binding site of the HLA-DR structure and/or HLA-DQ model.

The predicted nonameric core sequence in each epitope cluster of each of the potential autoantigens shown in Table 1 above are comprised within the sequences of SEQ ID NOs: 1-21 shown in FIGS. 10-14, as follows:

-   -   MOG: 34-56 [SEQ ID NO:1]; 67-114 [SEQ ID NO:2]; 3-27 [SEQ ID         NO:3]; 205-215 [SEQ ID NO:4].     -   MBP: 84-111 [SEQ ID NO:5]; 141-168 [SEQ ID NO:6]; 12-42 [SEQ ID         NO:7].     -   OSP: 42-73 [SEQ ID NO:8]; 98-109 [SEQ ID NO:9]; 187-206[SEQ ID         NO:10]; 192-206 [SEQ ID NO:11]; 20-33 [SEQ ID NO:12]; 129-145         [SEQ ID NO:13].     -   MOBP: 15-33 [SEQ ID NO:14]; 55-90 [SEQ ID NO:15]; 156-172 [SEQ         ID NO:16].     -   PLP: 103-150 [SEQ ID NO:17]; 177-203 [SEQ ID NO:18]; 218-240         [SEQ ID NO:19]; 38-52 [SEQ ID NO:20]; 264-276 [SEQ ID NO:21].

These nonameric core sequences which fit into the MS-relevant HLA-DR/DQ molecule and are flanked by 2-5 amino acids at their N- and C-termini are considered to represent potential epitopes associated with MS. The unaltered peptides of the invention encompassing these predicted epitopes for each target autoantigen have the sequences of SEQ ID NOs: 22-33, 10 and 34-47 as follows:

-   -   MOG: 37-58 [SEQ ID NO:22]; 65-95 [SEQ ID NO:23]; 7-32 [SEQ ID         NO:24]; 202-218 [SEQ ID NO:25].     -   MBP: 82-103 [SEQ ID NO:26]; 136-156 [SEQ ID NO:27]; 148-170 [SEQ         ID NO:28]; 7-29 [SEQ ID NO:29]; 25-45 [SEQ ID NO:30].     -   OSP: 38-64 [SEQ ID NO:31]; 48-77 [SEQ ID NO:32]; 94-112 [SEQ ID         NO:33]; 187-206 [SEQ ID NO:10]; 17-38 [SEQ ID NO:34]; 124-150         [SEQ ID NO:35].     -   MOBP: 13-38 [SEQ ID NO:36]; 54-80 [SEQ ID NO:37]; 72-89 [SEQ ID         NO:38]; 156-174 [SEQ ID NO:39].     -   LP: 102-122 [SEQ ID NO:40]; 120-150 [SEQ ID NO:41]; 173-200 [SEQ         ID NO:42]; 190-208 [SEQ ID NO:43]; 185-206 [SEQ ID NO:44];         213-243 [SEQ ID NO:45]; 35-57 [SEQ ID NO:46]; 261-276 [SEQ ID         NO:47].

In some of the peptides above, serine (S) residues were replaced by cysteine (C) residues.

C2. The Altered Peptide Ligands (APLs) for MS

Also encompassed by the present invention are immune-specific approaches directed to the potential protective and/or curative effect of analogs of immunodominant encephalitogenic epitopes where TCR contact residues have been manipulated, and which do not stimulate T cell clonal proliferation, i.e. “altered peptide ligands” (APLs).

The specificity of TCR recognition is conferred by only some residues of the peptide presented by MHC class II molecules (Sette et al., 1987; Gautam et al., 1992). To define these TCR contact residues, peptide analogs of specific antigens have been generated in a variety of antigen systems by introducing single amino acid substitutions, and tested for their capacity to stimulate activation events in the T cells specific for the relevant antigenic peptide. Data resulting from such analysis have demonstrated that some of the interactions with the TCR are more critical than others. Thus, the “primary TCR contact site” is defined as the amino acid focused on most intently by all T cells in a population specific for the same antigen, and this amino acid appeared to be the most critical residue of the determinant to the TCR binding. Secondary residues are defined as the other TCR contact sites, which are unique to each TCR in the population, and are assumed to play a lesser role in the overall interaction between TCR and ligand (Evavold et al., 1994; Sloan-Lancaster and Allen, 1996).

Analogs of immunogenic peptides in which the TCR contact sites have been manipulated have been termed “altered peptide ligands” (APLs). These peptides do not stimulate T cell clonal proliferation, but they do bind to the MHC molecules with similar affinities as the unaltered immunogenic peptide, inducing some but not all TCR-mediated effector functions.

The ability of APLs to induce different signaling events has been shown to result in several possible processes of peripheral T cell modulation, anergy and/or induction of Th2-type cytokine secretion and/or antagonistic inhibition of proliferation to the stimulatory peptide and/or inhibition of Th1-type cytokine secretion. Such attributes make APLs very attractive for the design of potential therapeutic approaches to autoimmune diseases.

Epitope-directed, altered peptide ligand (APL)-mediated, downregulation of pathogenic T-cell autoreactivities has been proven by several laboratories to be an effective strategy for immune-specific modulation of EAE. In EAE induced with MBP or PLP, the crucial contact residues involved in the interaction of the TCR wih the encephalitogenic epitope have been identified. APLs whereby these crucial TCR contact residues were altered by substitution with Ala or other amino acids were shown to be highly effective in inhibiting EAE induced with the relevant native MBP or PLP peptide. Similar observations were made with our designed MOG APLs as described hereinafter.

In MS, the human MBP 83-99 region (ENPVVHFFKNIVTPRTP) (comprised within SEQ ID NO: 26) is one of the major immunodominant MBP epitopes recognized in the context of DR2, the MHC class II haplotype most associated with MS. The TCR contact residues for this epitope have been defined with F-89 apparently representing a primary TCR contact residue, while H-88 and K-91 apparently serve as secondary TCR contact residues (Wucherpfenning et al., 1994; Windhagen et al., 1995; Singh et al., 1999; Kozovska et al., 1998). In EAE, an APL of human MBP 87-99, substituted with Ala at K-91, blocked the disease, apparently via induction of cytokine shift from Th1 to Th2 in the relevant T-cells (Gaur et al., 1997).

An in vitro analysis of the effect of MBP 83-99 APLs on DR2- and DR4-restricted MBP-specific T-cell clones isolated from MS patients revealed that, although the CDR3 motifs of the T-cell clones were diverse, the TCR contact residues within MBP 83-99 were highly conserved, with F-90 and K-91 being critical TCR contact points for both DR2- and DR-4-restricted T-cell clones (Kozovska et al., 1998). Although APLs with Ala substitutions at F-90 or K-91 abolished the positive signalling event in the majority of DR2- and DR4-restricted T-cell clones tested, not all clones could be inhibited by either of these APLs; APLs doubly substituted at these positions, however, abolished responses by all T-cell clones tested (Kozovska et al., 1998). In a further study, deviation of cytokine profile from Th0 to Th2 by T-cell clones in the presence of MBP 83-99 APLs substituted with Ala at the secondary TCR contact residue H-88 was described (Singh et al., 1999).

Data from in vitro analyses of the effect of MBP83-99 APLs on MS patient-derived T-cell clones, together with in vivo analyses with similar APLs in EAE, have led to clinical trials in MS patients. The results of two such phase II clinical trials with subcutaneously injected APLs containing substitutions at the same positions, i.e. positions 83, 84, 89 and 91, were recently reported (Kappos et al., 2000; Bielekova et al., 2000). These two early phase clinical trials, however, were halted prematurely because of adverse allergic reactions (indicating an overly active Th2 response) and, in one case, of exacerbation of MS (Nature Medicine 2000 October 6(10):1167-1182).

It is now well accepted that MBP is not the only potential primary target antigen in MS and that multiple autoreactivities, implicated in the initiation and/or perpetuation of the disease, can occur. In both studies above, it is suggested that therapeutic benefits could be obtained through “bystander” suppression effect (Bielekova et al., 2000; Kappos et al., 2000), whereby targeting of one myelin epitope may induce the production of anti-inflammatory Th2 cytokines, via immune deviation of the pathogenic T-cells and/or induction of Th2-secreting APL-specific T-cells. Recent studies in EAE suggest, however, that targeting the deleterious T-cell reactivity against a single epitope is unlikely to be sufficiently effective in treating a disease associated with multiple autoreactivities.

In the present invention, computer modeling of the binding mode of the selected epitope clusters to HLA-DR/DQ molecules most prominently associated with MS was used to predict for each epitope cluster the nonameric core sequences with preferred binding mode to the HLA-DR/DQ, and which were seen to fit into the MHC binding site of the HLA-DR structure and/or HLA-DQ model.

The predicted nonameric core sequences in each epitope cluster of each of the potential primary target antigens MOG, MBP, OSP, MOBP, and PLP are comprised within the peptides of SEQ ID NOs: 1-21, and are shown in FIGS. 10-14 (broken line below the sequence for the MS-associated HLA-DR, and full line above the sequence for the MS-associated HLA-DQ).

The nonameric core sequences which fit into the MS-relevant HLA-DR/DQ molecule and are flanked by 2-5 amino acids at their N- and C-termini were considered to represent potential epitopes associated with MS. The peptides encompassing these predicted epitopes for each target autoantigen MOG, MBP, OSP, MOBP, and PLP are represented by SEQ ID NOs: 22-33, 10 and 34-47.

For each of the peptides of SEQ ID NOs: 22-33, 10 and 34-47, the TCR contact residues were predicted according to the predicted core nonameric sequence binding to the MS-relevant HLA-DR/DQ molecules. For each autoantigen, the peptides encompassing predicted epitopes were modified in their TCR contact residues by replacement of one or more of said TCR contact residues by alanine (Ala), but substitutions by other suitable amino acids is also encompassed by the invention.

The selection of the TCR contact residues to be substituted took into consideration the overlapping potential epitopes within the clusters, so that autoreactivity against as many overlapping epitopes would be antagonized without interfering with their MHC binding residues or turning some epitopes to super-agonists.

Examples of altered peptides of the invention are represented by the SEQ ID NOs: 48-86, that correspond to the peptides of SEQ ID NOs: 22-33, 10 and 34-47, respectively, but in which one or more TCR contact amino acid residues have been replaced by Ala.

The resulting altered peptides of the invention derived from the autoantigens associated with MS are as follows:

-   -   MOG-AL peptides A-F (SEQ ID NOs: 48-53, respectively) in FIG. 15         and SEQ ID NOs: 232-233;     -   MBP-AL peptides A-H (SEQ ID NOs: 54-61, respectively) in FIG.         16;     -   OSP-AL peptides A-E, E′, F,G (SEQ ID NOs: 62-69, respectively)in         FIG. 17;     -   MOBP-AL peptides A-D (SEQ ID NOs: 70-73, respectively) in FIG.         18;     -   PLP-AL peptides A, A′,B-L (SEQ ID NOs: 74-86, respectively) in         FIG. 19.

The underlined in these sequences in FIGS. 18-19 are alanine (A) substitutions of the residues representing predicted TCR contact residues within the core sequence predicted to bind to MS-associated HLA class II.

Epitopes appropriately altered in their TCR contact residues, when appropriately administered, are expected to immunomodulate the potentially pathogenic T-cell response against the epitope with minimal risk of exacerbating the pathogenic T-cell response.

For the design of the APLs, for each selected epitope cluster of each autoantigen, computer modeling was used with the purpose to:

-   -   (i) identify the nonameric sequence(s) with preferred binding         mode to HLA-DR/DQ molecules most prominently associated with MS.         Briefly, the crystal structure of the DR molecule         (DRA*0101/DRB1*15011), deposited in the Protein Data Bank (PDB         code 1b×2), was used to characterize the binding preferences of         this MHC molecule. These were represented through a 9×20 matrix         in which every entry is a number quantifying the preference of a         given amino acid (20 possibilities) to be placed in a given         position along the bound peptide (9 possibilities). The         structure of the DQ molecule (DQA1*0102/DQB1*0602) is not known,         and was modeled according to related structures (PDB codesliak,         2seb), using the MSI modeling software package (MSI Inc.,         San-Diego, Calif.), in particular the Homology and Discover         modules. A 9×20 binding preference matrix was also constructed         for this molecule. The binding matrices were used in a computer         program in which a 9 amino-acid moving window is applied to the         sequence of each epitope cluster of each autoantigen and a         binding score is calculated for each 9 amino-acid sequence. This         score is estimated as the sum of the binding preferences from         the appropriate 9×20 matrix. One of our MHC structures is         modeled (DQA1*0102/DQB1*0602) and hence less reliable than an         experimental structure. Therefore, in order not to miss possible         epitopes, the binding preference matrices must not be too         strict. Sequences with binding scores above average were modeled         in analogy to MHC class II-bound peptides in known structures         (DR, I-A^(k)), and energy-minimized. Of these, sequences which         were seen to fit into the MHC binding site were taken as         representing possible HLA-binding epitopes.     -   (ii) Identify crucial TCR contact residues. Studies on crystal         structures of peptide-NFC class II complexes have shown that the         amino acids at positions 1, 4, 6 and 9 of the nonameric core         interacting with the MHC groove are the MHC-binding residues.         Amino acids at the other positions, i.e. positions 2, 3, 5, 7         and 8, are directed towards the TCR. It has been experimentally         demonstrated that, of these non MHC-binding residues, amino         acids at positions 2 and 5 are often the most important TCR         contact residues, with position 5 often representing the primary         TCR contact residue within the core nonameric sequence.         Accordingly, the non MHC-binding residues, preferably at         position 5 and/or 2, of the predicted nonameric sequence with         preferred binding mode to HLA-DR or -DQ were substituted with         Ala. However, substitution of these residues with other amino         acids is also envisaged.

FIGS. 10-14 show the nonameric sequences within the selected epitope clusters of each autoantigen, with binding score above average which were also found to fit into the MHC binding site of HLA-DR structure or of HLA-DQ models. Nonameric sequences indicated by broken lines (below sequence) were taken as representing HLA-DR binding epitopes; nonameric sequences indicated by full lines (above sequence) were taken as representing HLA-DQ binding epitopes. As can be seen in these figures, some regions within a selected epitope cluster may contain overlapping HLA-DR and/or HLA-DQ binding epitopes. Hence, alteration of one potential TCR contact residue for one epitope may not antagonize the autoimmune T-cell reactivity against another overlapping epitope. Alternatively, it may interfere with the MHC binding of another overlapping epitope, thus preventing the possibility to antagonize the potential pathogenic T-cell reactivity against this epitope(s). Another possibility may be that alteration of a TCR contact residue of one epitope may affect the overlapping epitope, turning it into a super-agonist rather than an antagonist. These possibilities have been taken into consideration in the selection of the TCR contact residues to be altered for each epitope cluster. Thus, for a region containing overlapping HLA-DR and/or HLA-DQ epitopes, several TCR contact residues may need to be altered, necessitating replicate sequences with substitutions at different positions (for example, see SEQ ID NOs: 48-86). The bolded and underlined amino acids in each epitope cluster shown in FIGS. 10-14, are the TCR contact residues determined to be substituted according to the above criteria.

The present invention thus provides a synthetic peptide comprising amino acid sequences of at least one IEC of at least one human autoantigen related to MS, said synthetic peptide being selected from:

-   -   (i) an unaltered synthetic peptide comprising at least one         nonameric core sequence which fits into the MS-relevant         HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N-         and C-termini, the resulting IEC being capable of stimulating         human T cells, wherein the peptides are those of SEQ ID NOs: 10         and 22-47; and     -   (ii) an altered synthetic peptide comprising at least one         nonameric core sequence which fits into the MS-relevant         HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its         N-and C-termini, in which sequence one to three T-cell receptor         (TCR) contact amino acid residues are substituted by a suitable         amino acid such as Ala, the resulting IEC altered in the TCR         residue being capable of immunomodulating the potentially         pathogenic T-cell response against the epitope without risk of         exacerbation, excluding altered peptides derived from the MBP         83-99 and PLP 139-151 sequences.

Altered peptides derived from the MBP 83-99 sequence are disclosed for example in U.S. Pat. No. 6,251,396 (altered MBP 83-99), U.S. Pat. No. 6,329,499 (altered MBP 86-99) and U.S. Pat. No. 6,369,033 (altered MBP 87-99). Altered PLP 139-151 peptides are disclosed by Hafler, 1996 and Young, 2002.

The IEC is derived from at least one human autoantigen related to MS such as MOG, MBP, OSP, MOBP, PLP, and MAG.

Examples of altered synthetic peptides of the invention include: (i) a peptide containing MOG epitope(s) in which one to three TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 48-53; (ii) a peptide containing MBP epitope(s) in which 1-3 TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 54-61; (iii) a peptide containing OSP epitope(s) in which 1-4 TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 62-69; (iv) a peptide containing MOBP epitope(s) in which 1-3 TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 70-73; and (v) a peptide containing PLP epitope(s) in which 1-3 TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 74-86.

D. The Synthetic Genes for MS

The present invention further provides a synthetic gene comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS or coding for at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said IECs have amino acid sequences selected from the sequences of the unaltered and/or altered peptides of the invention, for example the peptides of SEQ ID NOs: 10 and 22-86.

The synthetic genes comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS are herein in the specification and claims identified by a designation including the letters “sh” (standing for “synthetic human”) followed by the abbreviation of the autoantigen: shMOG, shMP, shOSP, shMOBP, and shPLP genes (FIGS. 1-5). When the IECs have amino acid sequences of the altered peptides, the letters “AL”(the abbreviation for “altered ligand”) follow the abbreviation of the autoantigen: shMOG-AL, shMBP-AL, shOSP-AL, shMOBP-AL, and shPLP-AL genes (FIGS. 15-19).

In one embodiment, the synthetic gene comprises nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS, wherein said IECs have amino acid sequences selected from the sequences of the unaltered peptides of the invention of SEQ ID NOs: 10 and 22-47. Examples are the shMOG (SEQ ID NO:102), shMBP (SEQ ID NO:114), shOSP (SEQ ID NO:128), shMOBP (SEQ ID NO:138), and shPLP (SEQ ID NO:154) genes comprising also restriction endonuclease sites as depicted in FIGS. 1-5, respectively.

In another embodiment, the synthetic gene comprises nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS, wherein said IECs have amino acid sequences selected from the sequences of the altered peptides of the invention, for example of SEQ ID NOs: 48-86. Examples are the shMOG-AL (SEQ ID NO:87), shMBP-AL (SEQ ID NO:88), shOSP-AL (SEQ ID NO:89), shMOBP-AL (SEQ ID NO:90), and shPLP-AL (SEQ ID NO:91) genes comprising also restriction endonuclease sites as depicted in FIGS. 15-19, respectively. For each autoantigen, the amino acid sequences representing epitope clusters with altered TCR contact residues (A, B, C . . . ) were aligned with or without addition of a linker comprised of glycine and serine (GS).

In a further embodiment, the synthetic gene is a multitarget autoantigen gene comprising nucleotide sequences coding for at least one IEC of at least two, preferably 3 or 4, more preferably 5 or more different autoantigens related to MS, and analogs thereof, wherein said IECs have amino acid sequences selected from the sequences of the unaltered and/or altered peptides of the invention.

The synthetic multitarget autoantigen genes comprising nucleotide sequences coding for at least one IEC of at least two different autoantigens related to MS, are herein in the specification and claims identified by a designation including an initial capital Y followed by the abbreviation MS for multiple sclerosis, the letter P and either a small letter “e”, when the IECs have amino acid sequences of the unaltered peptides of the invention, for example of SEQ ID NOs: 10 and 22-47, or the letters “AL”, when the IECs have amino acid sequences of the altered peptides of the invention, for example of SEQ ID NOs: 48-86. The synthetic multitarget autoantigen genes preferably comprise a high number of preferred IECs of the selected autoantigens, preferably of MOG, MBP, OSP, MOBP and PLP.

Examples of multitarget autoantigen genes wherein the IECs have amino acid sequences of the unaltered peptides of the invention of SEQ ID NOs: 10 and 22-47, are the genes herein designated Y-MSPe (SEQ ID NO: 225) and ΔY-MSPe (SEQ ID NO: 227), represented by the constructs of FIGS. 6 and 7, respectively.

Examples of multitarget autoantigen genes wherein the IECs have amino acid sequences of the altered peptides of the invention of SEQ ID NOs: 48-86, are the genes herein designated Y-MSP-AL (SEQ ID NO: 92) and ΔY-MSP-AL (SEQ ID NO: 93), represented by the constructs of FIGS. 20 and 21, respectively.

Synthetic genes and multitarget autoantigen genes wherein the IECs have amino acid sequences of the altered peptides of the invention constitute a preferred embodiment of the invention and include:

-   -   (i) a synthetic gene comprising nucleotide sequences coding for         at least two, preferably all the six MOG amino acid sequences of         the SEQ ID NOs: 48-53, each of them carrying 1-3 alanine         substitutions, more preferably the shMOG-AL gene of SEQ ID NO:         87 depicted by the construct of FIG. 15;     -   (ii) a synthetic gene comprising nucleotide sequences coding for         at least two, preferably all the eight MBP amino acid sequences         of the SEQ ID NOs: 54-60, each of them carrying 1-3 alanine         substitutions, more preferably the shMBP-AL gene of SEQ ID NO:         88 depicted by the construct of FIG. 16;     -   (iii) a synthetic gene comprising nucleotide sequences coding         for at least two, preferably all the eight OSP amino acid         sequences of the SEQ ID NOs: 61-69, each of them carrying 1-3         alanine substitutions, more preferably the shOSP-AL gene of SEQ         ID NO: 89 depicted by the construct of FIG. 17;     -   (iv) a synthetic gene comprising nucleotide sequences coding for         at least two, preferably all the four of the MOBP amino acid         sequences of the SEQ ID NOs: 70-73, each of them carrying 1-3         alanine substitutions, more preferably the shMOBP-AL gene of SEQ         ID NO: 90 depicted by the construct of FIG. 18;     -   (v) a synthetic gene comprising nucleotide sequences coding for         at least two, preferably all the 13 of the PLP amino acid         sequences of the SEQ ID NOs: 74-86, each of them carrying 1-3         alanine substitutions, more preferably the shPLP-AL gene of SEQ         ID NO: 91 depicted by the construct of FIG. 19;     -   (vi) a synthetic multitarget autoantigen gene comprising the         nucleotide sequences of at least two of the synthetic genes of         (i)-(v) above, preferably comprising all said sequences, more         preferably the Y-MSP-AL gene of SEQ ID NO: 92 depicted by the         construct of FIG. 20; and     -   (vii) a synthetic multitarget autoantigen gene being the         truncated form of (vi), comprising the nucleotide sequences of         at least two synthetic genes selected from the group consisting         of: (a) a gene comprising nucleotide sequences coding for the         MOG-AL amino acid sequences of the SEQ ID NOs: 48-51; (b) a gene         comprising nucleotide sequences coding for the MBP-AL amino acid         sequences of the SEQ ID NOs: 54-59; (c) a gene comprising         nucleotide sequences coding for the OSP-AL amino acid sequences         of the SEQ ID NOs: 62-67; (d) a gene comprising nucleotide         sequences coding for the MOBP-AL amino acid sequences of the SEQ         ID NOs: 70-73; and (e) a gene comprising nucleotide sequences         coding for the PLP-AL amino acid sequences of the SEQ ID NOs:         74-84; preferably the ΔY-MSP-AL gene of SEQ ID NO: 93 depicted         by the construct of FIG. 21.         E. Preparation of the Synthetic Genes of the Invention

With a knowledge of the autoantigens and immunogenic epitopic clusters associated with MS, the skilled person may prepare the synthetic genes encoding the IECs of the appropriate autoantigens by standard recombinant techniques. The synthetic genes may contain the coding sequences for the IECs in any particular order, for example, the coding regions for all the clusters from each autoantigen may be grouped together or, alternatively, IECs from different autoantigens may be organized randomly along the synthetic genes. Preferably, the former arrangement prevails and each cluster coding region may be separated from the next by 3, 6, 9 etc. nucleotides or possibly by a restriction site as shown in FIGS. 1-7 and FIGS. 15-21. Any spacing sequences may be inserted in between the coding regions provided that the single open reading frame for the totality of cluster coding regions is retained. In a preferred embodiment, the synthetic gene codes for at least one, preferably 2-3, immunogenic epitopic clusters specific for each of at least two autoantigens, though it may be more preferable to include 4 or 5 or possibly more clusters from each autoantigen. For the constructs of FIGS. 15-19, for each autoantigen, the amino acid sequences representing epitope clusters with altered TCR contact residues (A, B, C . . . ) were aligned with or without addition of a linker comprised of glycine and serine (GS).

General method for construction, amplification and cloning the synthetic genes Synthetic genes coding for the IECs of autoantigens related to MS are obtained stepwise. A synthetic gene for each autoantigen encoding the selected IECs arranged tandemly is prepared by PCR overlap extension using overlapping synthetic oligonucleotides encoding the IECs. Briefly, 60-70 nucleotide long oligonucleotides representing codons of the amino acid residues of the selected IECs are synthesized. The oligonucleotides overlap at their 5′ and/or 3′ ends by 18 nucleotides which are complementary to their neighboring oligonucleotides. Specific restriction endonuclease sites are included in the first and the last oligonucleotide to facilitate cloning, as well as to enable in frame ligation to neighboring synthetic genes (see below for the construction of the Y-MSPe and Y-MSP-AL genes).

The DNA sequence corresponding to the aligned, linked clusters was modified where necessary, to include alterations not resulting in amino acid changes to neutralize possibly problematic restriction endonuclease sites (RES), to minimize formation of DNA secondary structures or to “bacterize” codons, as well as alterations leading to substitution of cysteine for serine in order to increase the solubility of the expressed protein. DNA sequences corresponding to RES were added to enable cloning and expression, as well as to allow in frame ligation to neighbouring mini-genes.

A template for the synthetic gene is generated by mixing the relevant oligonucleotides (each 75 pmol) in Taq DNA polymerase buffer (40 μl final volume) containing dNTPs (RO181; MBI Fermentas AB, Vilnius, Lithuania) at a final concentration of 0.2 mM each and a mixture of 0.2 U Vent DNA polymerase and 0.2 U Taq DNA polymerase (AB-0192; Advanced Biotechnologies, Surrey, UK). After denaturation (94° C., 1 min) and annealing of the oligonucleotides through their complementary ends (55° C., 2 min), PCR overlap extension is carried out at 72° C. for 5 min, and the resulting template (4 μl) is amplified by PCR at standard conditions for 30 cycles, using the relevant oligonucleotides as 5′ and 3′ reverse primers. The amplified PCR product of the expected size is eluted from agarose gel and directly cloned into a T vector (pGEM-T, A3600; Promega Corp., Madison, Wis., USA). The constructed synthetic gene is cleaved out from the pGEM-T/synthetic gene plasmid and subcloned into the bacterial expression vector pRSET (V351-20; Invitrogen, San Diego, Calif., USA) via NheI and BglII, 3′ to its 6xHis tag, using standard molecular biology techniques. DNA sequence analysis is performed using the pRSET-specific primers to confirm the synthetic gene DNA sequence as an open reading frame with the ATG of the pRSET expression vector.

Other available technologies of molecular biology can be used by a person skilled in the art to prepare the synthetic genes of the invention.

EXAMPLE 1 Construction of the shMOG Gene

The shMOG gene (FIG. 1) was constructed using for template generation the primers designated MOG.p1 (SEQ ID NO: 94), MOG.p2 (reverse=rev), (SEQ ID NO: 95), MOG.p3 (SEQ ID NO: 96), MOG.p4 (rev) (SEQ ID NO: 97), MOG.p5 (SEQ ID NO: 98), MOG.p6 (rev) (SEQ ID NO: 99), and for amplification the 5′ and 3′ reverse primers MOG.p1a (SEQ ID NO: 100), and MOG.p6a (rev) (SEQ ID NO: 101). The shMOG DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 102 and SEQ ID NO: 103, respectively.

EXAMPLE 2 Construction of the shMBP Gene

The shMBP gene (FIG. 2) was constructed using for template generation the following primers: MBP.p1 (SEQ ID NO: 104), MBP.p2(rev), (SEQ ID NO: 105), MBP.p3 (SEQ ID NO: 106), MBP.p4(rev) (SEQ ID NO: 107), MBP.p5 (SEQ ID NO: 108), MBP.p6(rev) (SEQ ID NO: 109), MBP.p7 (SEQ ID NO: 110), MBP.p8(rev) (SEQ ID NO: 111), and for amplification the 5′ and 3′ reverse primers MBP.p1a (SEQ ID NO: 112), and MBP.p8a(rev) (SEQ ID NO: 113). The shMBP DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 114 and SEQ ID NO: 115, respectively.

EXAMPLE 3 Construction of the shOSP Gene

The shOSP gene (FIG. 3) was constructed using for template generation the following primers: OSP.p1 (SEQ ID NO: 116), OSP.p2 (rev) (SEQ ID NO: 117), OSP.p3 (SEQ ID NO: 118), OSP.p4 (rev) (SEQ ID NO: 119), OSP.p5 (SEQ ID NO: 120), OSP.p6 (rev) (SEQ ID NO: 121), OSP.p7 (SEQ ID NO: 122), OSP.p8 (rev) (SEQ ID NO: 123), OSP.p9 (SEQ ID NO: 124), OSP.p10 (rev) (SEQ ID NO: 125), and for amplification the 5′ and 3′ reverse primers OSP.p1a (SEQ ID NO: 126), and OSP.p10a (rev) (SEQ ID NO: 127). The shOSP DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 128 and SEQ ID NO: 129, respectively.

EXAMPLE 4 Construction of the shMOBP Gene

The shMOBP gene (FIG. 4) was constructed using for template generation the following primers: MOBP.p1 (SEQ ID NO: 130), MOBP.p2 (rev) (SEQ ID NO: 131), MOBP.p3 (SEQ ID NO: 132), MOBP.p4 (rev) (SEQ ID NO: 133), MOBP.p5 (SEQ ID NO: 134), MOBP.p6 (rev) (SEQ ID NO: 135), and for amplification the 5′ and 3′ reverse primers MOBP.p1a (SEQ ID NO: 136), and MOBP.p6a (rev) (SEQ ID NO: 137). The shMOBP DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 138 and SEQ ID NO: 139, respectively.

EXAMPLE 5 Construction of the shPLP Gene

The shPLP gene (FIG. 5) was constructed using for template generation the following primers: PLP.p1 (SEQ ID NO: 140), PLP.p2 (rev) (SEQ ID NO: 141), PLP.p3 (SEQ ID NO: 142), PLP.p4 (rev) (SEQ ID NO: 143), PLP.p5 (SEQ ID NO: 144), PLP.p6 (rev) (SEQ ID NO: 145), PLP.p7 (SEQ ID NO: 146), PLP.p8 (rev) (SEQ ID NO: 147), PLP.p9 (SEQ ID NO: 148), PLP.p10 (rev) (SEQ ID NO: 149), PLP.p11 (SEQ ID NO: 150), PLP.p12 (rev) (SEQ ID NO: 151), and for amplification the 5′ and 3′ reverse primers PLP.p1a (SEQ ID NO: 152), and PLP.p12a (rev) (SEQ ID NO: 153). The shPLP DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 154 and SEQ ID NO: 155, respectively.

EXAMPLE 6 Construction of the shMOG-AL Gene

The shMOG-AL gene (FIG. 15) was constructed using for template generation the following primers: MOG.AL.p1 (SEQ ID NO: 156), MOG.AL.p2 (rev) (SEQ ID NO: 157), MOG.AL.p3 (SEQ ID NO: 158), MOG.AL.p4 (rev) (SEQ ID NO: 159), MOG.AL.p5 (SEQ ID NO: 160), MOG.AL.p6 (rev) (SEQ ID NO: 161), MOG.AL.p7 (SEQ ID NO: 162), MOG.AL.p8 (rev) (SEQ ID NO: 163), and for amplification the 5′ and 3′ reverse primers MOG.AL.p1a (SEQ ID NO: 164), and MOG.AL.p8a (rev) (SEQ ID NO: 165). The shMOG-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 87 and SEQ ID NO: 166, respectively.

EXAMPLE 7 Construction of the shMBP-AL Gene

The shMBP-AL gene (FIG. 16) was constructed using for template generation the following primers: MBP.AL.p1 (SEQ ID NO: 167), MBP.AL.p2 (rev) (SEQ ID NO: 168), MBP.AL.p3 (SEQ ID NO: 169), MBP.AL.p4 (rev) (SEQ ID NO: 170), MBP.AL.p5 (SEQ ID NO: 171), MBP.AL.p6 (rev) (SEQ ID NO: 172), MBP.AL.p7 (SEQ ID NO: 173), MBP.AL.p8 (rev) (SEQ ID NO: 174), MBP.AL.p9 (SEQ ID NO: 175), MBP.AL.p10 (rev) (SEQ ID NO: 176), and for amplification the 5′ and 3′ reverse primers MBP.AL.p1a (SEQ ID NO: 177), and MBP.AL.p10a (rev) (SEQ ID NO: 178). The shMBP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 88 and SEQ ID NO: 179, respectively.

EXAMPLE 8 Construction of the shOSP-AL Gene

The shOSP-AL gene (FIG. 17) was constructed using for template generation the following primers: OSP.AL.p1 (SEQ ID NO: 180), OSP.AL.p2 (rev) (SEQ ID NO: 181), OSP.AL.p3 (SEQ ID NO: 182), OSP.AL.p4 (rev) (SEQ ID NO: 183), OSP.AL.p5 (SEQ ID NO: 184), OSP.AL.p6 (rev) (SEQ ID NO: 185), OSP.AL.p7 (SEQ ID NO: 186), OSP.AL.p8 (rev) (SEQ ID NO: 187), OSP.AL.p9 (SEQ ID NO: 188), OSP.AL.p10 (rev) (SEQ ID NO: 189), OSP.AL.p11 (SEQ ID NO: 190), OSP.AL.p12 (rev) (SEQ ID NO: 191), and for amplification the 5′ and 3′ reverse primers OSP.AL.p1a (SEQ ID NO: 192), and OSP.AL.p12a (rev) (SEQ ID NO: 193). The shOSP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 89 and SEQ ID NO: 194, respectively.

EXAMPLE 9 Construction of the shMOBP-AL Gene

The shMOBP-AL gene (FIG. 18) was constructed using for template generation the following primers: MOBP.AL.p1 (SEQ ID NO: 195), MOBP.AL.p2 (rev) (SEQ ID NO: 196), MOBP.AL.p3 (SEQ ID NO: 197), MOBP.AL.p4 (rev) (SEQ ID NO: 198), MOBP.AL.p5 (SEQ ID NO: 199), MOBP.AL.p6 (rev) (SEQ ID NO: 200), and for amplification the 5′ and 3′ reverse primers MOBP.AL.p1a (SEQ ID NO: 201), and MOBP.AL.p6a (rev) (SEQ ID NO: 202). The shMOBP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 90 and SEQ ID NO: 203, respectively.

EXAMPLE 10 Construction of the shPLP-AL Gene

The shPLP-AL gene (FIG. 19) was constructed using for template generation the following primers: PLP.AL.p1 (SEQ ID NO: 204), PLP.AL.p2 (rev) (SEQ ID NO: 205), PLP.AL.p3 (SEQ ID NO: 206), PLP.AL.p4 (rev) (SEQ ID NO: 207), PLP.AL.p5 (SEQ ID NO: 208), PLP.AL.p6 (rev) (SEQ ID NO: 209), PLP.AL.p7 (SEQ ID NO: 210), PLP.AL.p8 (rev) (SEQ ID NO: 211), LP.AL.p9 (SEQ ID NO: 212), PLP.AL.p10 (rev) (SEQ ID NO: 213), PLP.AL.p11 (SEQ ID NO: 214), PLP.AL.p12 (rev) (SEQ ID NO: 215), PLP.AL.p13 (SEQ ID NO: 216), PLP.AL.p14 (rev) (SEQ ID NO: 217), PLP.AL.p15 (SEQ ID NO: 218), PLP.AL.p16 (rev) (SEQ ID NO: 219), PLP.AL.p17 (SEQ ID NO: 220), PLP.AL.p18 (rev) (SEQ ID NO: 221), and for amplification the 5′ and 3′ reverse primers PLP.AL.p1a (SEQ ID NO: 222), and PLP.AL.p18a (rev) (SEQ ID NO: 223). The shPLP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 91 and SEQ ID NO: 224, respectively.

EXAMPLE 11 Construction of the Y-MSPe Gene

A synthetic multitarget autoantigen gene of the invention encoding randomly organized EECs of several autoantigens is prepared by ligating together the resulting synthetic genes for each autoantigen in one open reading frame to form the desired multiple gene.

Briefly, for construction of the Y-MSPe gene, the shMOG, shMBP, shOSP, shMOBP and shPLP genes of Examples 1-5 above are ligated sequentially via specific endonuclease restriction sites which have been incorporated to allow their ligation in one open reading frame as shown in the construct depicted in FIG. 6. To this end, the pGEM-T/shMOG is cleaved at the BamHI and HindIII sites, the shMBP gene is excised from pGEM-T/shMBP with BamHI and SpeI, the shOSP gene is excised from pGEM-T/shOSP with SpeI and PstI, the shMOBP gene is excised from pGEM-T/shMOBP with PstI and XhoI, and the shPLP gene is excised from pGEM-T/shPLP with XhoI and HindIII. The DNA fragments of the right sizes are eluted from agarose gel, cleaned and sequential ligations are carried out to link the shMBP gene to the shMOG gene via their BamHI sites, the shOSP gene to the shMBP gene via their SpeI sites, the shMOBP gene to the shOSP gene via their PstI sites, and the shPLP gene to the shMOBP gene via their XhoI sites and to the pGEM-T/shMOG via their HindIII sites, as depicted in FIG. 6. The resulting ligated DNA fragment comprising the five synthetic genes representing Y-MSPe is then subcloned into the pRSET bacterial expression vector (Invitrogen), 3′ to its 6xHis tag, via the NheI and HindIII restriction sites. DNA sequence analysis is performed using the pRSET-specific primers to confirm the Y-MSPe DNA sequence as an open reading frame with the ATG of the pRSET expression vector. The Y-MSPe DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 225 and SEQ ID NO: 226, respectively.

EXAMPLE 12 Construction of the ΔY -MSPe Gene

For construction of the ΔY-MSPe gene, the MS-related truncated Y-MSPe gene coding only for preferred epitopes likely to be more frequently recognized in disease on each of the autoantigens selected, is prepared as follows: the pGEM-T/shPLP is cleaved at BamHI and BclI and the small BamHI/BclI fragment is removed by gel electrophoresis. The pGEM-T/shPLP is then religated via the compatible BamHI/BclI sites to generate the pGEM-T/shΔPLP. The XhoI/HindIII DNA fragment comprising the shΔPLP gene is excised out from the pGEM-T/shΔPLP and ligated into the pRSET/Y-MSPe digested with XhoI and HindIII to replace the shPLP gene. The resulting plasmid (pRSET/Y-MSPe with shPLP) is then digested with NheI and HindIII and the excised DNA fragment (Y-MSPe with shΔPLP) is sequentially cleaved and religated via the compatible restriction sites BglII and BamHI, followed by XbaI and SpeI, followed by NsiI and PstI, as depicted in FIG. 7. The resulting DNA fragment comprising the shΔ symthetic genes is cloned back into the pRSET vector via the NheI and HindIII sites. The resulting plasmid is the pRSET/ΔY-MSPe (ΔY-MSPe). DNA sequence analysis is performed using the pRSET-specific primers to confirm the ΔY-MSPe DNA sequence as an open reading frame with the ATG of the pRSET expression vector. The ΔY-MSPe DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 227 and SEQ ID NO: 228, respectively.

EXAMPLE 12 Construction of the Y-MSP-AL and ΔY-MSP-AL Genes

The Y-MSP-AL and ΔY-MSP-AL genes are constructed in exactly the same manner as described in Examples 10-11 above, according to the schemes depicted in FIGS. 20 and 21, respectively.

The Y-MSP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 92 and SEQ ID NO: 229, respectively.

The ΔY-MSP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 93 and SEQ ID NO: 230, respectively.

F. The Polypeptides of the Invention and Their Preparation

The invention further provides a synthetic polypeptide comprising amino acid sequences of at least two IECs of a sole autoantigen related to MS or at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said amino acid sequences are selected from the sequences of the unaltered and/or altered peptides of the invention.

In one embodiment, the polypeptide comprises the unaltered amino acid sequences of at least two IECs of a sole autoantigen related to MS consisting of the polypeptides shMOG (SEQ ID NO:103), shMBP (SEQ ID NO:115), shOSP (SEQ ID NO:129), shMOBP (SEQ ID NO:139), and shPLP (SEQ ID NO:155).

In another embodiment, the polypeptide comprises the unaltered amino acid sequences of at least one IEC of at least two different autoantigens related to MS, preferably the polypeptides Y-MSPe (SEQ ID NO:226) and ΔY-MSPe (SEQ ID NO:228).

In a preferred embodiment, the polypeptides comprise the sequences of the altered peptides of the invention and include, for example:

-   -   (i) a polypeptide comprising at least two, preferably all the         six MOG-AL amino acid sequences of the SEQ ID NOs: 48-53, each         of them carrying 1-3 alanine substitutions, preferably         containing all the six sequences, more preferably the         polypeptide of the amino acid sequence of SEQ ID NO: 166;     -   (ii) a polypeptide comprising at least two, preferably all the         eight MBP-AL amino acid sequences of the SEQ ID NOs: 54-61, each         of them carrying 1-3 alanine substitutions, preferably         containing all the eight sequences, more preferably the         polypeptide of the amino acid sequence of SEQ ID NO: 179;     -   (iii) a polypeptide comprising at least two, preferably all the         eight OSP-AL amino acid sequences of the SEQ ID NOs: 62-69, each         of them carrying 1-4 alanine substitutions, preferably         containing all the eight sequences, more preferably the         polypeptide of the amino acid sequence of SEQ ID NO: 194;     -   (iv) a polypeptide comprising at least two, preferably all the         four of the MOBP-AL amino acid sequences of the SEQ ID NOs:         70-73, each of them carrying 1-3 alanine substitutions,         preferably comprising the four sequences, more preferably the         polypeptide of the amino acid sequence of SEQ ID NO: 203;     -   (v) a polypeptide comprising at least two, preferably all the 13         of the PLP-AL amino acid sequences of the SEQ ID NOs: 74-86,         each of them carrying 1-3 alanine substitutions, more preferably         the polypeptide of the amino acid sequence of SEQ ID NO: 224;     -   (vi) a polypeptide comprising: (a) at least two, preferably all         the six MOG-AL amino acid sequences of the SEQ ID NOs:         48-53; (b) at least two, preferably all the eight MBP-AL amino         acid sequences of the SEQ ID NOs: 54-61; (c) at least two,         preferably all the eight OSP-AL amino acid sequences of the SEQ         ID NOs: 62-69; (d) at least two, preferably all the four of the         MOBP-AL amino acid sequences of the SEQ ID NOs: 70-73; and (e)         at least two, preferably all the 13 of the PLP-AL amino acid         sequences of the SEQ ID NOs: 74-86, each of the amino acid         sequences carrying 1-3 alanine substitutions, preferably         comprising all said sequences (a)-(e), more preferably the         polypeptide of the amino acid sequence of SEQ ID NO: 229; and     -   (vii) a polypeptide being the truncated form of (vi),         comprising: (a) the MOG amino acid sequences of the SEQ ID NOs:         48-51; (b) the MBP amino acid sequences of the SEQ ID NOs:         54-59; (c) the OSP amino acid sequences of the SEQ ID NOs:         62-67; (d) the MOBP amino acid sequences of the SEQ ID NOs:         70-73; and (e) the PLP amino acid sequences of the SEQ ID NOs:         74-84; each of the amino acid sequences carrying 1-3 alanine         substitutions, preferably the polypeptide of the amino acid         sequence of SEQ ID NO: 230.

For the preparation of the polypeptides, the synthetic genes of the present invention can be incorporated into expression vector that may be, for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said synthetic gene and optionally a regulator of the promoter. The recombinant expression vector may then be used to transform or transfect suitable host cells such as bacterial cells, e.g. E. coli cells, or eukaryotic cells such as yeast, insect or preferably, mammalian cells, to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression of the polypeptide. The expressed polypeptide is then recovered by extraction from the host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption, and isolated by protein purification methods known in the art, such as metal chelate chromatography, HPLC, antibody-affinity chromatography etc.

EXAMPLE 13 Expression of the Protein Products of the Synthetic Genes

The synthetic gene of any one of Examples 1-12 above is cleaved out from the pGEM-T/synthetic gene plasmid and subcloned into the bacterial expression vector pRSET (V351-20; Invitrogen, San Diego, Calif., USA) via suitable restriction sites such as NheI and BglII or NheI and HindIII, 3′ to its 6xIis tag, using standard molecular biology techniques. DNA sequence analysis is performed using the pRSET-specific primers to confirm the synthetic gene DNA sequence as an open reading frame with the ATG of the pRSET expression vector.

The pRSET/synthetic gene is then transformed into Escherichia coli host (BL21-DE3), and protein expression is induced by isopropyl β-D-thio-galactopyranoside (IPTG) (RO392; MBI Fermentas AB). After expression is observed, the expressed protein product is isolated under denaturing conditions (8 M urea) by metal chelate affinity chromatography on Ni²⁺ nitriloacetic acid (NTA) agarose (30230; Qiagen Inc., Valencia, Calif., USA) according to the manufacturer's protocol. Fractions containing the isolated protein, as evidenced by SDS-PAGE, are pooled and subjected to reducing conditions with β-mercaptoethanol. The protein is diluted to 50-100 μg/ml in 8 M urea and allowed to refold by dialysis against gradually decreasing concentrations of urea (8-0 M). Any aggregated protein is removed by centrifugation.

The synthetic gene may also be expressed in a mammalian expression vector as described in WO 01/31037. Thus, the synthetic gene DNA is cleaved out from the pGEM-T/synthetic gene plasmid with EcoRI and NotI and subcloned into the mammalian expression vector pCDNA 3.1. The pCDNA 3.1/synthetic gene is transfected into mammalian cells, e.g. NIH3T3 mouse fibroblasts, CHO or any other suitable mammalian cells, and the expressed protein product is isolated and examined by standard molecular biology techniques.

G. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions for multiepitope-directed immunomodulation of MS comprising a pharmaceutically acceptable carrier and a mixture of two or more unaltered and/or altered peptides of the invention, or a synthetic gene of the invention or a polypeptide which is a protein product of a synthetic gene of the invention.

The synthetic genes and their protein products may be useful for concomitant immunomodulation of potentially pathogenic autoreactivities against different or multiple epitopes of a single or several target antigens in MS, via DNA vaccination or via tolerogenic administration of the protein product of the synthetic gene.

Thus, the present invention further relates to a pharmaceutical composition for multiepitope-directed immunomodulation of MS comprising a pharmaceutically acceptable carrier and an agent selected from:

-   -   (i) a mixture of at least two synthetic peptides, each peptide         comprising amino acid sequences of at least one immunogenic         epitope cluster (IEC) of at least one human autoantigen related         to multiple sclerosis (MS), said peptide being selected from an         unaltered synthetic peptide comprising at least one nonameric         core sequence which fits into the MS-relevant HLA-DR/DQ molecule         and is flanked by 2-5 amino acids at its N- and C-termini, the         resulting IEC being capable of stimulating human T cells;     -   (ii) a mixture of at least two synthetic peptides, each peptide         comprising amino acid sequences of at least one immunogenic         epitope cluster (IEC) of at least one human autoantigen related         to multiple sclerosis (MS), said peptide being selected from an         altered synthetic peptide comprising at least one nonameric core         sequence which fits into the MS-relevant HLA-DR/DQ molecule and         is flanked by 2-5 amino acids at its N- and C-termini, in which         sequence one to three T-cell receptor (TCR) contact amino acid         residues are substituted by a suitable amino acid such as Ala,         the resulting IEC altered in the TCR residue being capable of         immunomodulating the potentially pathogenic T-cell response         against the epitope without risk of exacerbation;     -   (iii) a synthetic gene of the invention; and     -   (iv) a polypeptide of the invention.

The formulation of the pharmaceutical composition of the invention comprising a mixture of peptides will depend upon the route of administration but typically they can be formulated for topical, parenteral, intramuscular, intravenous, intra-peritoneal, intranasal inhalation, lung inhalation, or intradermal administration. The polypeptide may be administered in an injectable form by mixing with a pharmaceutically acceptable vehicle for an injectable formulation.

The pharmaceutically acceptable carrier or diluent may be, for example, sterile isotonic saline solutions, or other isotonic solutions such as phosphate-buffered saline. Additional ingredients of the formulations include suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Typically, the physician will determine the actual dosage, which will be most suitable for an individual patient, and it will vary with the age, weight and response of the particular patient.

For some applications, preferably the compositions are administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents.

The compositions as well as the polypeptides alone can also be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. In this case, the compositions will comprise a suitable carrier or diluent. For parenteral administration, the compositions are best used in the form of a sterile aqueous solution, which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.

The present invention also provides pharmaceutical compositions for the treatment of MS comprising at least one synthetic gene of the invention to be administered via “naked DNA” vaccination or together with a suitable gene delivery vehicle. The gene delivery vehicle may be a non-viral vehicle such as cationic liposomes or a cationic lipid vehicle, or a viral vehicle such as adenovirus vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector or a baculoviral vector. The gene can be, for example, integrated in a retroviral expression vector.

When injected in a soluble tolerogenic route (s.c., i.v. or i.p.) or administered by oral or nasal application, the composition of the invention is expected to downregulate the potentially pathogenic autoimmune responses in MS. Alternatively, when administered as a naked DNA constructed into an appropriate mamalian expression vector, the synthetic gene of the invention can be effective in vaccinating against the disease.

H. Diagnostic Compositions

The unaltered and altered peptides as well as the polypeptides comprising sequences of the unaltered and altered peptides of the invention can also be used for diagnosis and/or for monitoring the progression of MS by measuring the levels of immunoactivation of T-cells specific for the autoantigen(s) associated with the disease. T-cell responses to the peptides or polypeptides are likely to be higher in patients than in control individuals, and measurement of such responses by blood cells or serum can be used as a diagnostic/monitoring tool.

The levels of T-cell autoreactivity to the peptides or polypeptides are measured by incubating PBLs isolated from peripheral blood in the presence of the relevant peptides or polypeptides, and monitoring the activation of the reactive T-cells by detection of T-cell proliferation, cytokine release and expression of cytokine receptors and other activation-associated cell surface markers. Such assays of T-cell activation are well known to those of skill in the art.

EXAMPLE 14 MOG APLs Substituted at Crucial TCR Contact Residues 41 and 44 Suppress EAE Induced with MOG 37-52

The altered peptides of the invention were tested for their biological activity in relevance to MS, using EAE as a model system.

For EAE induction, female 2-3 months old C3H.SW mice (Jackson Laboratory, Bar harbor, Maine, USA) were immunized with MOG 37-52 (150 μg emulsified with CFA supplemented with 300 μg Mycobacterium tuberculosis). On days 5, 7, 9 and 12 after encephalitogenic challenge, the mice were injected intravenously (i.v.) with an aqueous solution of MOG 37-52 (SEQ ID NO: 231), MOG 37-A44-52 (SEQ ID NO: 232), or MOG 37-Q41-52 (SEQ ID NO: 233), (400 μg in PBS), or with a combination of MOG 37-A44-52+MOG 37-Q41-52, or with PBS alone. Mice were followed and scored daily for clinical effects on a scale of 0-6. The results in FIG. 8 show that treatment with MOG 37-A44-52 and/or MOG 37-Q41-52 led to a marked decrease in severity and incidence of EAE induced with the native MOG 37-52, an observation associated with a markedly reduced response ex vivo to MOG 37-52 by lymph node cells isolated from treated mice.

For EAE induced in H-2^(b) haplotype mice (C3H.SW, C57BL) by MOG 35-55 (SEQ ID NO: 234), we have defined residues 41 and 44 as crucial TCR contact residues for the interaction between the encephalitogenic nonameric core sequence MOG 40-48, found by molecular modelling studies to represent the preferred binding mode for MOG 35-55 to IA^(b), and the TCR of H-2^(b) MOG-specific T-cells. APLs with non-conservative substitutions at either or both of these residues elicited T-cell responses which were not encephalitogenic (not shown). In vitro, these APLs inhibited the proliferation of T-cell clones to MOG 37-52, the minimal optimal stimulatory peptide. Pilot studies of the cytokine profile of APL-specific T-cell lines suggested that the APL wherein F44 is substituted by A44 (MOG 37-A44-52) elicits Th2 T-cells (not shown), an observation commensurate with the non-encephalitogenicity of this APL, and which could play a role in immunomodulation of MOG-induced EAE. MOG35-55-specific T-cell line was raised from MOG35-55-immunized C3H.SW mice as described (Mendel et al., 1996). Cytokine assays were performed by ELISA according to PharMingen standard protocols.

Possible mechanisms by which the relevant APLs may inhibit MOG 37-52-reactive T-cell clones in vitro were tested by analyzing their effect on TCR signaling, as well as on cytokine secretion by the clones. The results indicated that MOG 37-A44-52 alone induces partial TCR signaling in the T-cell clones, suggesting that it may exert its effect via partial agonist as well as antagonist activities, which could result in anergy of the T-cells (not shown).

EXAMPLE 15 Inhibition of EAE Associated with Multiple Pathogenic Autoreactivities Requires Multiepitope-directed Immunomodulation

Y-MSPb, the purified protein product of the synthetic Y-MSPb gene, disclosed in WO 01-31037, designed to encode tandemly arranged disease-relevant epitopes clusters of the encephalitogenic proteins MBP, PLP, MOG, and MOBP, was tested for its immunomodulatory effect on EAE associated with multiple pathogenic autoreactivities, upon tolerogenic administration, in the same way as described for Y-MSPa in WO 01-31037.

Line T-cells were selected in vitro with Y-MSPb from lymph node cells (LNCs) of (C3H.SW×SJL/J)F₁ mice immunized with Y-MSPb (containing encephalitogenic epitopes of MOG, MBP, PLP and MOBP) in CFA, and were analyzed after primary response (left panel) and after four cycles of selection (right panel) for their proliferative response to Y-MSPb, phMOG 34-56, phMOG 1-22, shMOG/E, PLP 139-151, shPLP/E, MBP 87-101 and shMBP/E (10 μ/ml). The proliferative response to purified protein derivative (PPD) (5 μ/ml) was analyzed as a measure of specificity. The results are shown in FIG. 9A. The line T cells are directed against each of the myelin autoantigens.

FIG. 9B shows that EAE associated with multiple pathogenic autoreactivities induced by Y-MSPb-reactive line T-cells (2×10−⁶ cells, i.v., on day 0) is fully abrogated by tolerogenic administration of Y-MSPb in PBS given daily intraperitoneally (i.p.) from the day of T-cell transfer. Targeting the T-cells reacting against only one of the epitopes, MOG 37-52 or PLP139-151, recognized by the multireactive Y-MSPb-elicited line T-cells, has no significant effect on disease incidence or severity, while dual targeting with a combination of MOG 37-52 and PLP139-151 (indicated as MOG+PLP) has only a marginal effect on disease severity.

The development of EAE associated with multiple autoreactivities and induced by transfer of T-cells reactive with defined epitopes of MBP, PLP, MOG and MOBP (FIG. 9A) could also be totally suppressed by daily i.v. administration of the purified protein product of the Y-MSPb gene (FIG. 9B). The relevant PLP or MOG peptides administered singly according to the same regimen had no effect on disease development, while a combination of MOG+PLP only marginally decreased disease severity (FIG. 9B). These data strongly emphasize the necessity to neutralize as many as possible of the relevant multiple autoreactivities for effective immunomodulation of autoimmune diseases associated with a multiplicity of potential primary target antigens. In view of eventual extrapolation to therapeutic approaches for MS, concomitant multiepitope-directed immunomodulation via non-stimulatory, non-encephalitogenic APLs is likely to be a more effective, safer approach.

In the same way, the multitarget autoantigen genes of the invention Y-MSPe, ΔY-MSPe, and preferably the Y-MSP-AL and ΔY-MSP-AL genes can be tested, and their ability to immunomodulate potentially pathogenic autoreactivities against multiple epitopes of several antigens associated with MS, can be shown.

References

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1. An isolated synthetic peptide comprising amino acid sequences of at least one immunogenic epitope cluster (IEC) of at least one human autoantigen related to multiple sclerosis (MS), said synthetic peptide being selected from the group consisting of: (i) an unaltered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N- and C-termini, the resulting IEC being capable of stimulating human T cells, wherein the peptides are those of SEQ ID NOs: 10 and 22-47; and (ii) an altered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N-and C-termini, in which sequence one to three T-cell receptor (TCR) contact amino acid residues are substituted by a suitable amino acid, the resulting IEC altered in the TCR residue being capable of immunomodulating the potentially pathogenic T-cell response against the epitope without risk of exacerbation, excluding altered peptides derived from the MBP 83-99 and PLP 139-151 sequences.
 2. An isolated synthetic peptide according to claim 1, wherein the autoantigen related to MS is selected from the group consisting of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin-oligodendrocytic basic protein (MOBP), oligodendrocyte-specific protein (OSP) and proteolipid protein (PLP).
 3. An isolated synthetic peptide according to claim 2, selected from the group of unaltered peptides consisting of MOG peptides of SEQ ID NOs:22-25; MBP peptides of SEQ ID NOs:26-30; OSP peptides of SEQ ID NOs:10, 31-35; MOBP peptides of SEQ ID NOs:36-39; and PLP peptides of SEQ ID NOs:40-47.
 4. An isolated synthetic peptide according to claim 2, wherein said peptide is an altered peptide derived from a human autoantigen selected from the group consisting of ,MOG, MBP, OSP, MOBP and PLP, and said suitable amino acid is alanine (Ala).
 5. An altered synthetic peptide according to claim 4, selected from the group consisting of the MOG-AL peptides of SEQ ID NOs: 48-53 and 232-233; the MBP-AL peptides of SEQ ID NOs: 54-61; the OSP-AL peptides of SEQ ID NOs: 62-69; the MOBP-AL peptides of SEQ ID NOs: 70-73; and the PLP-AL peptides of SEQ ID NOs: 74-86.
 6. An isolated synthetic polypeptide comprising amino acid sequences of at least two IECs of a sole autoantigen related to MS or at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said amino acid sequences are selected from the group consisting of: (i) an unaltered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N- and C-termini, the resulting IEC being capable of stimulating human T cells, wherein the peptides are those of SEQ ID NOs: 10 and 22-47; and (ii) an altered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N-and C-termini, in which sequence one to three T-cell receptor (TCR) contact amino acid residues are substituted by a suitable amino acid, the resulting IEC altered in the TCR residue being capable of immunomodulating the potentially pathogenic T-cell response against the epitope without risk of exacerbation, excluding altered peptides derived from the MBP 83-99 and PLP 139-151 sequences.
 7. An isolated synthetic polypeptide comprising amino acid sequences of at least two IECs of a sole autoantigen related to MS or at least one IEC of at least two different autoantigens related to MS, wherein said sequences are selected from the group consisting of the unaltered synthetic MOG peptides of SEQ ID NOs:22-25; MBP peptides of SEQ ID NOs:26-30; OSP peptides of SEQ ID NOs:10, 31-35; MOBP peptides of SEQ ID NOs:36-39; and PLP peptides of SEQ ID NOs:40-47.
 8. An isolated synthetic polypeptide according to claim 7 selected from the group consisting of the polypeptides shMOG (SEQ ID NO:103), shMBP (SEQ ID NO:115), shOSP (SEQ ID NO:129), shMOBP (SEQ ID NO:139), and shPLP (SEQ ID NO:155).
 9. An isolated synthetic polypeptide according to claim 7, the polypeptide comprising the unaltered amino acid sequences of at least one IEC of at least two different autoantigens related to MS.
 10. An isolated synthetic polypeptide according to claim 9 selected from the group consisting of the polypeptides Y-MSPe (SEQ ID NO:226) and ΔY-MSPe (SEQ ID NO:228).
 11. An isolated synthetic polypeptide comprising amino acid sequences of at least two IECs of a sole autoantigen related to MS or at least one IEC of at least two different autoantigens related to MS, wherein said autoantigen related to MS is selected from the group consisting of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin-oligodendrocytic basic protein (MOBP), oligodendrocyte-specific protein (OSP) and proteolipid protein (PLP), and wherein said synthetic polypeptide comprises sequences of synthetic altered peptides comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N-and C-termini, in which sequence one to three T-cell receptor (TCR) contact amino acid residues are substituted by a suitable amino acid, the resulting IEC altered in the TCR residue being capable of immunomodulating the potentially pathogenic T-cell response against the epitope without risk of exacerbation, excluding altered peptides derived from the MBP 83-99 and PLP 139-151 sequences.
 12. An isolated synthetic polypeptide according to claim 11, wherein said suitable amino acid is alanine (Ala).
 13. An isolated synthetic polypeptide according to claim 12 comprising amino acid sequences of at least two IECs of a sole autoantigen related to MS or at least one IEC of at least two different autoantigens related to MS, wherein said sequences are selected from the group consisting of the altered synthetic MOG-AL peptides of SEQ ID NOs: 48-53 and 232-233; the MBP-AL peptides of SEQ ID NOs: 54-61; the OSP-AL peptides of SEQ ID NOs: 62-69; the MOBP-AL peptides of SEQ ID NOs: 70-73; and the PLP-AL peptides of SEQ ID NOs: 74-86.
 14. An isolated synthetic polypeptide according to claim 13 selected from the group consisting of the polypeptides shMOG-AL (SEQ ID NO:166), shMBP-AL (SEQ ID NO:179), shOSP (SEQ ID NO:194), shMOBP (SEQ ID NO:203), and shPLP (SEQ ID NO:224).
 15. An isolated synthetic polypeptide according to claim 14, the polypeptide comprising the altered amino acid sequences of at least one IEC of at least two different autoantigens related to MS.
 16. An isolated synthetic polypeptide according to claim 15 selected from the group consisting of the polypeptides Y-MSP-AL (SEQ ID NO:229) and ΔY-MSPAL (SEQ ID NO:230).
 17. A pharmaceutical composition for immunomodulation and treatment of multiple sclerosis comprising a mixture of at least two peptides according to claim 1, and a pharmaceutically acceptable carrier.
 18. A pharmaceutical composition according to claim 17, wherein said mixture contains at least one peptide of at least two different autoantigens.
 19. A pharmaceutical composition according to claim 17, wherein said mixture contains at least two peptides of a sole autoantigen.
 20. A pharmaceutical composition for immunomodulation and treatment of multiple sclerosis comprising at least one polypeptide according to claim 6, and a pharmaceutically acceptable carrier.
 21. A diagnostic composition for the diagnosis and/or monitoring of progression of multiple sclerosis comprising a mixture of two or more peptides according to claim 1, and a pharmaceutically acceptable carrier.
 22. A synthetic gene comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS or coding for at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said IECs have amino acid sequences selected from the group consisting of the sequences of the unaltered and/or altered peptides according to claim
 1. 23. An isolated synthetic gene according to claim 22, wherein the autoantigen related to MS is selected from the group consisting of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin-oligodendrocytic basic protein (MOBP), oligodendrocyte-specific protein (OSP) and proteolipid protein (PLP).
 24. An isolated synthetic gene according to claim 23, wherein said IECs have amino acid sequences selected from the group of sequences of the unaltered peptides consisting of MOG peptides of SEQ ID NOs:22-25; MBP peptides of SEQ ID NOs:26-30; OSP peptides of SEQ ID NOs:10, 31-35; MOBP peptides of SEQ ID NOs:36-39; and PLP peptides of SEQ ID NOs:40-47.
 25. An isolated synthetic gene according to claim 24, selected from the group consisting of shMOG gene of SEQ ID NO:102; shMBP gene of SEQ ID NO:114; shOSP gene of SEQ ID NO:128; shMOBP gene of SEQ ID NO:138; and shPLP gene of SEQ ID NO:154.
 26. An isolated synthetic gene according to claim 23, wherein said IECs have amino acid sequences from altered synthetic peptides derived from a human autoantigen selected from the group consisting of MOG, MBP, OSP, MOBP and PLP, and said suitable amino acid is alanine (Ala).
 27. An isolated synthetic gene according to claim 26, wherein said altered peptides are selected from the group consisting of MOG-AL peptides of SEQ ID NOs: 48-53 and 232-233; the MBP-AL peptides of SEQ ID NOs: 54-61; the OSP-AL peptides of SEQ ID NOs: 62-69; the MOBP-AL peptides of SEQ ID NOs: 70-73; and the PLP-AL peptides of SEQ ID NOs: 74-86.
 28. An isolated synthetic gene according to claim 27, selected from the group consisting of the genes shMOG-AL (SEQ ID NO:87), shMBP-AL (SEQ ID NO:88), shOSP (SEQ ID NO:89), shMOBP (SEQ ID NO:90), and shPLP (SEQ ID NO:91).
 29. An isolated synthetic gene according to claim 26, comprising nucleotide sequences coding for altered amino acid sequences of at least one IEC of at least different autoantigens related to MS.
 30. An isolated synthetic gene according to claim 29 selected from the group consisting of the genes Y-MSP-AL (SEQ ID NO:92) and ΔY-MSPAL (SEQ ID NO:93).
 31. A pharmaceutical composition for multiepitope-directed immunomodulation of multiple sclerosis comprising a synthetic gene according to claim 22, and a pharmaceutically acceptable carrier.
 32. A diagnostic composition for the diagnosis and/or monitoring of progression of multiple sclerosis comprising a polypeptide according to claim 6 and a pharmaceutically acceptable carrier. 